The role of DNA integration in the HIV-1 life cycle has been well characterized. Following binding of HIV-1 to a sensitive cell, the viral and cellular membranes fuse and the viral core particle is released into the cytoplasm. There the viral genomic RNA is reverse transcribed, yielding a double stranded DNA copy of the viral RNA genome. Next, a complex of viral DNA and proteins--the "preintegration complex"--covalently attaches the viral cDNA to host DNA. The integration step is required for replication, as demonstrated for example by the finding that HIV derivatives containing lesions in the integrase protein are unable to replicate (Clavel et al., (1989) J. Virol. 63:1455-1459 and Shin et al., (1994) J. Virol. 68:1633-1642). Integration completes the formation of a provirus, which contains all the information necessary to direct the synthesis of the viral RNAs and proteins required for the formation of new virions (Goff, S. P. (1992) Annu. Rev. Genet. 26:527-544).
In vivo, integration of retroviral DNA specifically requires the viral-encoded integrase protein (Clavel, F. et al., (1989), J. Virol. 63:1455-1459), and DNA sites at each end of the unintegrated viral DNA (Colicelli, J. et al. (1985) Cell 42:573-580 and Panganiban, A. T. et al. (1983) Nature 306:155-160). Integrase protein is normally synthesized as a part of the gag-pol precursor, and is released from the carboxy-terminus of reverse transcriptase by the action of the viral protease.
Purified integrase protein is capable of catalyzing the formation of a covalent bond between a model viral DNA and a target DNA in vitro (Bushman, F. D. et al. (1991) Proc. Natl. Acad. Sci. USA 88:1339-1343, Bushman, F. D. et al. (1990) Science 249:1555-1558 and Craigie, R. et al. (1990) Cell 62:829-837). Data presented in the references establishes that integrase is not just a required cofactor, but instead is the true recombinase that joins viral DNA to host DNA.
In vivo, prior to integration, the blunt ended DNA product of reverse transcription is cleaved so as to remove two nucleotides from each 3' end. The recessed 3' ends are then joined to 5' ends of breaks made in the target DNA. The resulting integration intermediate is then processed, probably by host DNA repair enzymes, to complete the attachment of each viral 5' end to host DNA (FIG. 1a) (for a recent review see Goff, S. P. (1992) Annu. Rev. Genet. 26:527-544).
In reactions in vitro, purified integrase can cleave the 3' end of a model viral end-substrate and catalyze covalent integration of the recessed 3' end into a target DNA (FIG. 1b) (Bushman, F. D. et al. (1990) Science 249:1555-1558, Craigie, R. et al. (1990) Cell 62:829-837 and Katz, R. A. et al. (1990) Cell 63:87-95). The product of these reactions resembles the unrepaired intermediate generated during integration in vivo. There is little specificity, however, both in vivo and in vitro in the target DNA sequence in which integration occurs.
Despite safety concerns, retroviral vectors are currently a popular means for delivering DNA in gene therapy protocols. Attractive features include controllable cell type specificity of delivery, stable insertion of the delivered genes into the host genome, and stable maintenance of genes once integrated. Genes can either be delivered in replication competent viruses (i.e., a heterologous coding region in a viral genome) or, more commonly, as retroviral vectors. In retroviral vector systems, packaging cell lines are used that express the viral proteins from unpackagable RNAs. A DNA construct containing the gene of interest flanked by LTRs and a packaging signal sequence (.psi.) is introduced in the packaging cell line (see, e.g., Rosenberg, S. A., 1990, New Engl. J. Med., 323:570-578). Because the only packagable RNA is encoded by the vector sequences, the viral particles produced contain exclusively the therapeutic gene. Infection of a target cell then results in reverse transcription and integration of the therapeutic gene. As a result of these attractive features, many of the protocols so far approved for human gene therapy employ retroviral delivery systems (Morgan, R., 1993, BioPharm, 6(1):32-35).
Many studies in vertebrate systems establish that insertion of retroviral DNA can result in inactivation or ectopic activation of cellular genes, thereby causing diseases (for a recent review see Lee, Y. M. H., et al. (1990) J. Virol. 64:5958-5965). This represents a serious safety concern in using retroviral gene delivery systems in human systems. One well studied consequence of retroviral integration is activation of oncogenes. Several probable cases of activation of a human oncogene by insertion of HIV have been described (Shiramizu et al., 1994, Cancer Res., 54:2069-2072). Thus, methods for site-specifically controlling the location of integration of retroviral vectors are desired to overcome the prior art problem of insertional mutagenesis of the host genome.
In addition, researchers who identify important DNA-binding proteins by genetic assays also wish to identify the genes regulated by these proteins. Examples include the identification of regulatory genes associated with chromosomal breakpoints in tumors (e.g., Djabali, et al., 1992, Nature Genetics, 2:113-118; and Kinzler and Vogelstein, 1990, Mol. Cel. Biol., 10:634-642) and the discovery of regulatory genes associated with developmental defects in humans and other organisms. Thus, new in vitro methods for rapidly locating and isolating the genes controlled by DNA-binding proteins are desired.